During the third and last year of this research project the emphasis will be on the completion of our sequence studies on small BrCN-derived hemepeptides of P-450CAM, P-450LM-2, P-450LM-4, P-450scc and P-450llbeta. This work includes a detailed comparison of photocovalently labeled hemepeptides using the arylazido derivatives of typical substrates and common P-450 inhibitors in order to determine the corresponding contact residues. From this information we hope to learn the approximate position of the various substrates and inhibitors within the hemepeptides, relative to each other and relative to the heme group. Both the distance of a given label from the heme group and its spacial orientation relative to the heme iron will provide valuable insight into the conformation of the microenvironment including the heme and substrate binding sites. These results will, in turn, lead to a better understanding of the spectral shifts elicited by ligand binding and of the mode of action of P-450 hemeproteins. Since one SH group of P-450CAM is protected by substrate we will test a new covalent affinity approach using substrate derivatives which can readily interact with free SH groups. In extension of our comparative studies, P-450Rh fractions a, b and c, and P-450MEG as well as the P-450 hemeproteins involved in C-21 and 17 alpha hydroxylation of steroids in adrenocortical microsomes will be purified further, and then our procedure for generation of hemepeptides will be applied to these additional hemeproteins. Suitable substrate and inhibitor derivatives will be synthesized for specific photoaffinity labeling of the respective binding sites. Finally, we shall submit microsomal preparations from liver and adrenal cortex to our photo-affinity labeling procedure. This will permit us to study the number of types of P-450 hemeproteins accessible in the membrane and to determine the extent of specific label incorporation.